regulation of environmental responses in the pacific oyster, crassostrea gigas mackenzie gavery...

Regulation of environmental responses in the Pacific oyster,

Crassostrea gigas

Mackenzie Gavery

University of Washington

School of Aquatic and Fishery Sciences

Committee Meeting: April 15th, 2014

Agenda:

Research update

Proposed timeline

Environmental Response in Oysters

2009 2010 2011 2012 2013


2009 2010 2011 2012 2013

Cyclooxygenasecharacterization


2009 2010 2011 2012 2013

Gene expression: disease

resistance



2009 2010 2011 2012 2013

Whole transcriptome

Chapter I. CBP: Part D


resistance


Chapter I.RNA-Seq

HIGH IMPACTLOW IMPACT


742 novel contigs427 differentially expressed

Chapter I.RNA-Seq

Ultra-short read technology is a powerful tool for gene discovery and expression analysis in organisms with limited genomic resources


742 novel contigs427 differentially expressed

Chapter I.RNA-Seq


2009 2010 2011 2012 2013

Whole transcriptome


resistance




2009 2010 2011 2012 2013

Chapter II. BMC Genomics

Frontiers

Whole transcriptome

DNA methylation:single gene/in silico


resistance



DNA metabolism

cell cycle and proliferation

RNA metabolism

protein metabolism

death

other metabolic processes

cell organization and biogenesis

other biological processes

transport

stress response

developmental processes

cell-cell signaling

signal transduction

cell adhesion

0.45 0.50 0.55 0.60 0.65 0.70CpGO/E

Chapter II. in silico analysis‘h

ouse

keepin

g’

‘induci

ble

’

Predicted degree of DNA methylation

DNA metabolism

cell cycle and proliferation

RNA metabolism

protein metabolism

death

other metabolic processes

cell organization and biogenesis

other biological processes

transport

stress response

developmental processes

cell-cell signaling

signal transduction

cell adhesion

0.45 0.50 0.55 0.60 0.65 0.70CpGO/E

‘house

keepin

g’

‘induci

ble

’

DNA methylation detected by targeted bisulfite sequencing and methylation specific PCR

DNA methylation patterns are different between functionally distinct classes of genes

Chapter II. in silico analysis

Predicted degree of DNA methylation


2009 2010 2011 2012 2013

Frontiers

Whole transcriptome



resistance





2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring


resistance





2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring

transgenerational (5-aza pesticide)

EE2


resistance





2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring

bringing it all together! meth & expression


EE2


resistance





2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring


Ch III. PeerJtransgeneration

al (5-aza pesticide)

EE2


resistance





2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring


Chapter IV. Brief Func Gen


EE2


resistance




Ch III. PeerJ

Chapter III: whole methylome

genomic DNA

Approach

High-throughput bisulfite sequencing

genomic DNA

Approach



genomic DNA

Approach


RNA-Seq data


Gill tissue: > 2.5 million CG dinucleotides


scaffold 86 (Galaxy Trackster)

ex

CG

genes

exons

%methylation

0bp 200,000bp

100%

0%


ex

CG

genes

exons

%methylation

0bp 200,000bp

100%

0%

scaffold 86 (Galaxy Trackster)


Distribution in genomic elements


Distribution in genomic elements

Exon25%

Intron45%

Promoter4%

TE5%

Other22%


Relationship with expression


RNA-Seq data (Zhang et al., 2012)

Relationship with expression

Gene expression (Deciles)

DN

A m

eth

yla

tion/g

ene


Relationships with other gene specific attributes?


Relationships with other gene specific attributes?

Spatial

e.g. number of exons, length of mRNA

Gene expression (Zhang et al 2012):

Mean expression level across 9 tissues

Variation in expression between tissues (%CV)


PC1 (50.2%)

PC

2 (

26.1

%)


Chapter III: summary

unmethylatedmethylatedChapter III: summary

Gene function:

unmethylatedmethylatedChapter III: summary

Gene function:

unmethylated

induciblehousekeeping

methylatedChapter III: summary

Gene function:

Expression:

unmethylated

induciblehousekeeping


Gene function:

Expression:

unmethylated

inducible

low

housekeeping

high


Gene function:

Expression:

Tissue specific

expression:

unmethylated

inducible

low

housekeeping

high


Gene function:

Expression:

Tissue specific

expression:

unmethylated

inducible

low

housekeeping

high

methylated

variable conserved


Gene function:

Expression:

Tissue specific

expression:

unmethylated

inducible

low

housekeeping

high

methylated

variable conserved

A context dependent role of DNA methylation in bivalves (Gavery & Roberts 2014)

Chapter IV.


Summary of Chapters:I. Transcriptomic approaches to

understanding environmental response

II. First characterization of DNA methylation

in oysters (in silico analysis)

III. DNA methylation & gene expression:

whole genome analysis

IV. Perspective/Review: DNA methylation in

bivalves


2009 2010 2011 2012 2013

Frontiers

Whole transcriptome


Nanostring



EE2


resistance




Ch III. PeerJ



2009 2010 2011 2012 2013

BMC Genomics

Frontiers

Whole transcriptome


Nanostring



EE2


resistance


Authorea link


Ch III. PeerJ


https://www.authorea.com/users/6045/articles/6321

https://www.authorea.com/users/6045/articles/6321

Agenda:

Research update

Proposed timeline

Proposed Timeline:

Draft proposal to reading committee: April 21st

Proposed Timeline:

Mem

Day

Spring

End

Estrogen Experiment

Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?

Estrogen Experiment


EE2 (n=50/tank

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)

Day 35(n=15/tx)

Day 60(n=120/tx)

Estrogen Experiment


EE2 (n=50/tank

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)

Day 35(n=15/tx)

Day 60(n=120/tx)

7m / 3f

Estrogen Experiment


EE2 (n=50/tank)

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)

Day 35(n=15/tx)

Day 60(n=120/tx)

7m / 3f

10m / 5f - control

Estrogen Experiment


EE2 (n=50/tank)

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)

Day 35(n=15/tx)

Day 60(n=120/tx)

7m / 3f

10m / 5f - control6m / 7f / 1? – EE2

Estrogen Experiment


EE2 (n=50/tank)

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)

Day 35(n=15/tx)

Day 60(n=120/tx)

7m / 3f

10m / 5f - control6m / 7f / 1? – EE2

EE2 control0%

20%

40%

60%

80%

100%

30 40

6563

18 12

H

U

F

M

Estrogen Experiment


EE2 (n=50/tank)

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)

Day 35(n=15/tx)

Day 60(n=120/tx)

7m / 3f

10m / 5f - control6m / 7f / 1? – EE2

Estrogen Experiment


EE2 (n=50/tank

control (n=50/tank)

Day 0(n=10)

Day 7(n=15/tx)

Day 35(n=15/tx)

Day 60(n=120/tx)

7m / 3f

10m / 5f - control6m / 7f / 1? – EE2

Estrogen Experiment

Identifying differential methylation in day 7 females

Approach: MBD-Chip

Results:

45 differentially methylated regions

42 in genes, 3 upstream of genes

38 different genes identified

E.g.: CRHR, serotonin receptor, acetylcholine receptor, various DNA binding proteins

Next steps: validate DMRs by pyrosequencing

Appendices:Gene expression:

1. Characterization of cycloxygenase in C. gigas

2. Gene expression in disease resistant oysters

DNA methylation:

4. Nanostring report

5. Transgenerational Experimental Design

6. EE2 experiment: phenotypic data & DNA methylation results

Prostaglandins in oyster immunity

Prostaglandins in oyster immunity

Sequenced full length gene

Phylogenetic analysis

Tissue Distribution

Immune Response

Gene Expression in Disease Resistant oysters

Crassostrea virginica

Resistant and Naïve population

Immune Related Genes

Major Findings:

Resistant population appeared to be ‘primed’ with higher level of baseline expression of genes prior to exposure

Nanostring Project

Targeted methylation analysis

nCounter system

Methylation at 48 target genes

About 30% of the targets performed well

Major findings

Identified first differential methylation between tissue types

Transgenerational Experiment

Do methylation patterns persist across generations?

Expsure to either:

5-azacytidine

Vinclozolin

Spawned and raised larvae

Parents sampled: nanostring assay

Growout

Samples 18 months later (gametes)

Findings: size difference, no meth difference by Nanostring

regulation of environmental responses in the pacific oyster, crassostrea gigas mackenzie gavery...

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